EP4249500A2 - Peptide entities with antimicrobial activity against multi-drug resistant pathogens - Google Patents

Peptide entities with antimicrobial activity against multi-drug resistant pathogens Download PDF

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Publication number
EP4249500A2
EP4249500A2 EP21865361.6A EP21865361A EP4249500A2 EP 4249500 A2 EP4249500 A2 EP 4249500A2 EP 21865361 A EP21865361 A EP 21865361A EP 4249500 A2 EP4249500 A2 EP 4249500A2
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seq
group
peptide
gram
pharmaceutical composition
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German (de)
English (en)
French (fr)
Inventor
Vivian MONTERO-ALEJO
Rolando PERDOMO-MORALES
Amanda VÁZQUEZ-GONZÁLEZ
Hilda Elisa GARAY-PEREZ
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Centro de Investigacion y Desarrollo de Medicamentos CIDEM
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Centro de Investigacion y Desarrollo de Medicamentos CIDEM
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to new peptide entities, combinations, and pharmaceutical compositions comprising them; as well as their use in the manufacture of medicines and in methods of treating bacterial infections.
  • Antibiotic resistance occurs when germs like bacteria and fungi develop the ability to defeat or circumvent drugs designed to kill them. That means that the germs do not die and continue to grow. Infections caused by germs resistant to antibiotics are difficult and sometimes impossible, to treat. If we add to this that in most cases antibiotic-resistant infections require long hospital stays, additional medical follow-up visits, and expensive and toxic treatment alternatives, the complexity of the problem is even greater. Antibiotic resistance does not mean that the body is becoming resistant to antibiotics as occurs in resistance phenomena in other diseases; rather, the bacteria have become resistant to antibiotics designed to kill them. This problem is not solved by increasing the dose of antibiotics to be administered, which also represents an added risk of toxicity.
  • Antibiotic resistance is capable of affecting people at any stage of life, as well as the health, veterinary and agricultural industries, making it one of the most serious and urgent health problems in the world. Although the risk of resistant infections cannot be completely avoided, some people are at higher risk than others, for example, those with chronic diseases. If antibiotics lose their effectiveness, we lose the ability to treat infections and control threats to public health (https://www.cdc.gov/drugresistance/intl-activities/amr-challenge.html).
  • clinicians also rely on the ability to prevent infections through the use of antibiotics, for example in joint replacements, organ transplants, antiinflammatory therapy, cancer and in the treatment of chronic diseases such as diabetes, asthma, and rheumatoid arthritis.
  • microorganisms that are reported with the highest incidence of antibiotic resistance are Acinetobacter baumannii sp, resistant to carbapenems, Pseudomonas aeruginosa sp, resistant to carbapenems and Enterobacteriaceae spp, resistant to carbapenems and producing ESBL.
  • other microorganisms with an intermediate incidence are mentioned, such as Enterococcus faecium sp, resistant to vancomycin, and Staphylococcus aureus sp, resistant to methicillin.
  • AMPs antimicrobial peptides
  • the membrane lytic mechanism of natural AMPs is a potentially promising therapeutic alternative because resistance mechanisms rapidly emerge to specific drug targets [ Mourtado, R., et al., Design of stapled antimicrobial peptides that are stable, non-toxic and kill antibiotic-resistant bacteria in mice. Nature Biotechnology, 2019. 37 (10): p. 1186-1197 ].
  • the need for new external anti-infective agents is more urgent to combat infectious diseases, and specifically, those unresolved and/or caused by multi-drug-resistant microorganisms.
  • AMPs are selective towards negatively charged bacterial membranes such as bacteria, and their cytotoxicity is moderate towards eukaryotic organisms which present a neutral charge in their surface.
  • a distinctive element in the antibacterial mechanism of AMPs is their interaction with the cytoplasmic membrane, therefore charge and hydrophobicity are key properties for the development of the antimicrobial activity.
  • the mechanism of action generally involves the formation of lytic pores or destabilization of the membrane by the formation of peptide aggregates.
  • Other non-lytic mechanisms include cell depolarization, and translocation to the cytosol, and binding to intracellular targets (nucleic acids, enzymes, etc.) [ Hale, J.D. and R.E. Hancock, Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther, 2007. 5 (6): p. 951-9 .].
  • the present invention provides synthetic peptide sequences from the group comprising the sequences SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 with potential therapeutic applications in the prevention and treatment of microbial infections caused by multi-drug resistant bacteria.
  • the invention further provides a combination therapy to a patient with a bacterial infection to control the development of the multi-resistance phenomenon, which comprises the administration to the patient of at least 2 sequences selected from the group comprising SEQ ID NO. 1-3. And it also provides a combination therapy comprising the administration to the patient of a sequence selected from the group comprising SEQ ID NO. 1-3 with a beta-lactam antibiotic. And more specifically it provides this combination therapy where the beta-lactam antibiotic class are carbapenems.
  • Another object of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide selected from the group consisting of SEQ ID NO. 1-3 as an active ingredient and one or more pharmaceutically acceptable excipients.
  • Another object of the invention is a pharmaceutical composition that further comprises a second peptide selected from the group consisting of SEQ ID NO. 1-3 as an active ingredient.
  • Another object of the invention is a pharmaceutical composition comprising a peptide selected from the group consisting of SEQ ID NO. 1-3 and also a beta-lactam antibiotic. A particular embodiment of this object is where the antibiotic is meropenem.
  • the peptides described in the present invention can be used for the manufacture of drugs for the treatment of bacterial infections.
  • a distinctive aspect of the present invention is its use in the treatment of infections caused particularly by Gram-negative bacteria.
  • Particular forms of carrying out the invention are its therapeutic use in the treatment of infections caused by pathogenic strains belonging to the ESKAPE group, for example, Klebsiella pneumoneae sp, Acinetobacter baumannii sp, Pseudomona aeruginosa sp. and Escherichia coli sp, and more specifically, in clinical isolates of said species multi-resistant to conventional antibiotics.
  • Another particular aspect of the present invention is its use in the treatment of infections caused by Gram-positive bacteria.
  • Particular forms of carrying out the invention would be its use in the treatment of infections caused by pathogenic strains selected from the group of Enterococcus faecium sp or Staphylococcus aureus sp, and more specifically, in clinical isolates of said species multi-resistant to conventional antibiotics.
  • Another object of the present invention is a method of treatment or preventing of a microbial infection that comprises the administration of a peptide selected from the group consisting of SEQ ID No. 1-3, of any of the combinations or the pharmaceutical compositions.
  • a particular embodiment is where the infection is caused by a pathogenic strain belonging to the ESKAPE group.
  • the present invention provides an alternative based on new synthetic peptide compounds that combat pathogenic microorganisms of a bacterial nature, and that appears as multiresistant microorganisms to conventional antibiotics.
  • the chimeric hybrid structures designed from different families of natural peptides identified in the hemocytes of the lobster P. argus show antimicrobial activity against multiresistant antibiotic strains that include Enterococcus faecium sp., Staphylococcus aureus sp., Klebsiella pneumoneae sp., Acinetobacter baumannii sp., Pseudomonas aeruginosa sp. and Eccherichia coli sp.
  • the list of amino acid sequences that are selected from the group consisting of the sequences SEQ ID NO is shown. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3, or a peptide structure with at least 80% identity with SEQ ID NO. 1-3 is shown.
  • the selected sequences comprised in the group SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 are obtained by chemical synthesis using the solid phase peptide synthesis (SPPS) methodology. That said, in the primary structure of these sequences there are at least 6 basic amino acids corresponding to arginine or lysine that can occur in their L or D forms, and in a preferred embodiment the sequences obtained must contain at least four residues of arginine.
  • SPPS solid phase peptide synthesis
  • the present invention describes a combination therapy that includes at least one of the selected sequences comprised in the group SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3.
  • the peptides can be found in the same formulation or in different formulations that can be administered concomitantly or sequentially.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one synthetic peptide selected from the group consisting of SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 as an active ingredient and one or more pharmaceutically acceptable excipients.
  • the present invention provides the use of the sequences SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 in the prevention and/or treatment of microbial infections in a subject.
  • the peptide sequences of the present invention are broad-spectrum antibacterial because they have a lytic effect on Gram-negative and Gram-positive bacterial strains, which cause infections in humans and/or animals.
  • the present invention provides the use of said peptide sequences SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 are inhibitors of bacterial growth and with bactericidal activity against selected pathogenic microorganisms from the group Enterococcus faecium sp, Staphylococcus aureus sp, Klebsiella pneumoneae sp, Acinetobacter baumannii sp, Pseudomonas aeruginosa sp and Eccherichia coli sp.
  • the present invention provides the use of peptide sequences SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 as growth inhibitors of multi-resistant pathogenic microorganisms such as Enterococcus faecium sp, Staphylococcus aureus sp, Klebsiella pneumoneae sp, Acinetobacter baumannii sp, Pseudomonas aeruginosa sp, and Eccherichia coli sp. Consequently, all antibiotic multi-resistant microorganisms were sensitive to peptide compounds, presenting CL 90 values between 1.0 -8.0 ⁇ M in the kinetic microdilution method.
  • multi-resistant pathogenic microorganisms such as Enterococcus faecium sp, Staphylococcus aureus sp, Klebsiella pneumoneae sp, Acinetobacter baumannii sp, Pseudom
  • the present invention provides the use of peptide sequences SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 to obtain a 3-log decrease in the initial bacterial load in times less than 30 minutes. It is a fact that these provide rapidity to achieve the reduction of the bacterial load compared to the antibiotics meropenem and ciprofloxacin against the selected bacteria Klebsiella pneumoniae (ATCC 1003) and Pseudomona aeruginosa (ATCC 9027).
  • the peptide sequences SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3 show neutralizing activity of lipopolysaccharide or LPS, the main and toxic component of the outer membrane of Gram-negative bacteria.
  • the peptide sequences inhibit LPS-induced pro-inflammatory cytokine release in whole blood culture and inhibit the LPS response to the LAL (Limulus amebocyte lysate) reagent.
  • the invention further provides an embodiment that exemplifies the synergistic antibacterial effect obtained from the combinations of SEQ ID NO. 1- SEQ ID NO. 2; SEQ ID NO. 1- SEQ ID NO. 3 and SEQ ID NO. 2- SEQ ID NO. 3 in an in vitro culture of multi-antibiotic resistant strains of Klebsiella pneumoniae sp and Staphylococcus aureus sp from clinical isolates.
  • the present invention further provides a method of treatment of a subject diagnosed with a microbial infection, which comprises the administration of a therapeutically effective amount of one of the peptides selected from the group formed by SEQ ID NO. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3.
  • said infection is an infection caused by Gram-negative and Gram-positive bacteria multi-resistant to conventional antibiotics.
  • the present invention further provides that said treatment method guarantees a faster antibacterial response when the selected peptides of the sequences SEQ ID NO are combined. 1; SEQ ID NO. 2; and/or SEQ ID NO. 3, with the carbapenem antibiotic in an in vitro culture of Klebsiella pneumoniae sp and Staphylococcus aureus sp strains from the clinic.
  • a hoe was seeded in Mueller-Hinton liquid medium (MH) and allowed to grow at 37 °C. with orbital shaking at 180 rpm overnight. Subsequently, a working culture was prepared by inoculating 100 ⁇ L of growth in 5 mL of MH and incubated at 37oC for 2 or 3 hours. Growth stopped when the culture reached an optical density (OD) at 650nm between 0.45 and 0.55.
  • MH Mueller-Hinton liquid medium
  • the culture is considered to be at a concentration of 108 CFU/mL
  • the bacterial suspension was diluted to 10 6 CFU/mL in Phosphate Buffer Saline (PBS), which was used as a working culture.
  • PBS Phosphate Buffer Saline
  • the peptides were dissolved in sterile PB to the desired concentration, and 50 ⁇ L was applied to a sterile 96-well polypropylene plate (Eppendorf, Germany).
  • Table 1 Values expressed in lethal concentration of 90% of the bacterial culture (LC 90 ) of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 on multi-resistant strains (from the ESKAPE group) from clinical isolates. The values represent the range of concentrations obtained from independent experiments.
  • MDR clinical isolated LC 90 ⁇ M Gram-negative Gram-positive Peptides K. pneumoniae E. coli P. aeruginosa A. baumannii S . aureus E. faecium SEQ ID NO. 1 2-8 1-4 2-8 2-8 1-4 4-8 SEQ. ID. NO. 2 1-4 1-4 1-4 2-8 1-4 2-8 SEQ. ID. NO. 3 2-8 1-4 1-4 4-8 2-8 4-8 4-8;
  • the hemolytic activity of the peptides was evaluated on Wistar rat erythrocytes.
  • the drawn blood was supplemented with heparin in a collecting device (Monovette, Sarstedt, Germany).
  • the blood was centrifuged at 1000 ⁇ g for 10 minutes at 25 °C (Hettich, Germany), and the erythrocyte pellet was washed three successively with sterile saline.
  • Plasma-free erythrocytes were diluted in saline to a final concentration of 4% (v / v).
  • % H The percentage of hemolysis (% H) was calculated according to Equation 1, from two independent replications, where H 0% and H 100% correspond to the Abs540nm for the controls with PBS and Triton X-100, respectively, and H peptide at the value of Abs540nm obtained for each peptide concentration.
  • % H H peptide ⁇ H 0 % H 100 % ⁇ H 0 %
  • the cytotoxic effect of the peptides represented by SEQ ID NO.1-3 in Hep2 cells was determined.
  • 96-well plates seeded after 48 hours with a completely confluent cell monolayer (1 ⁇ 10 5 cells/well) were used.
  • the growth medium was removed from the plates and 100 ⁇ L of peptides dissolved in culture medium without SFBI were added per well at different concentrations from 6.25 ⁇ M to 100 ⁇ M.
  • Six wells were used per concentration of the peptide to be evaluated, and in the remaining wells (6) 100 ⁇ L of culture medium without SFBI were added, the latter constituted the cellular control.
  • the plates were incubated at 37 °C, in a 5% CO 2 atmosphere for 72 hours, and were observed daily to determine possible morphological changes in the cell monolayer indicative of cytotoxicity. After this time, cell viability was determined in all wells using the colorimetric assay based on the reduction of the MTT compound by active mitochondrial enzymes in living cells [ Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65 (1-2): p. 55-63 ] 10 ⁇ L of the MTT solution at a concentration of 5 mg/mL in PBS was added to each well.
  • AbsCT mean absorbance value of the cell cultures treated with the peptides. AbsCC: mean absorbance value of the cell controls considered as 100% cell viability. The assay was performed in duplicate for each peptide concentration. The figure shows the two variants of in vitro cytotoxicity assays of the peptides represented by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ. ID. NO. 3.
  • This method is based on the determination of the time needed by the peptides represented by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ. ID. NO. 3, and the antibiotics meropenem and ciprofloxacin, to achieve at least a 3-log (1000-fold) decrease in initial bacterial concentration CFUs.
  • Time-dependent death was evaluated against P. aeruginosa (ATCC 9027) and K. pneumoniae (ATCC 1003) bacterial strains as described in the literature [12], with minor modifications.
  • Working cultures of bacteria (1 ⁇ 105 CFU/mL) were exposed to all antimicrobials at the concentration of 2 ⁇ the MIC shown for the strain to be tested, in a final volume of 1 ml (v/v).
  • the inoculum in PBS (pH 7.4) without any antimicrobial was considered as a control.
  • all suspensions were incubated at 37 °C.
  • 100 ⁇ l aliquots of the culture were removed at different predetermined times (5, 10, 15, 20, 30, 60, 90, and 120 min), which were diluted 100 ⁇ in sterile PBS and spread on Mueller-Hinton agar plates. Plates were incubated for 24 hours at 37 ° C, and cell survival was determined by counting total colonies.
  • CFU versus time curves was constructed for each peptide compound and compared with the reference antibiotics. Bacteria death time is shorter for designed sequences than for conventional antibiotics used as susceptibility control for Gram-negative strains.
  • This method is based on the determination of pro-inflammatory cytokines released in a whole blood culture induced by agents that generate a pro-inflammatory response.
  • LPS was used as the main inducer of the pro-inflammatory response.
  • SEQ ID NO. 1 SEQ ID NO. 2 and SEQ. ID. NO. 3
  • the release of IL-6 cytokine was determined by an ELISA.
  • a whole blood culture was incubated in RPMI-1640 medium (Sigma, USA) with a series of dilutions of the peptides covering 0.05-50 nM in the presence and absence of fixed concentrations of LPS at 0.25 EU.
  • the designed sequences show an inhibitory activity of the expression of pro-inflammatory cytokines (IL-6) mediated by LPS in a whole blood culture.
  • IL-6 pro-inflammatory cytokines
  • the neutralizing activity of the peptides is represented by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ. ID. NO. 3 on bacterial endotoxins (LPS) using the LAL kinetic chromogenic assay (PYROCHROME ® ; ACC, USA). All pyrogen-free materials supplied by ACC, USA were used to carry out the test. The test conditions followed the manufacturer's instructions.
  • the peptides to be evaluated were tested in a series of 1:10 dilutions (v / v) from an initial concentration of 20 ⁇ M with a constant endotoxin challenge (LPS) of 0.5 EU / ml.
  • the different peptide concentrations plus the LPS challenge were incubated in a non-pyrogenic microplate (ACC, USA) at 37 °C for 5 minutes. Subsequently, the LAL reagent was added and the reaction kinetics were measured at 450 nm for 1 hour with 15-second intervals at 37 oC in a microplate spectrophotometer. The appearance of a yellow coloration was due to the release of p-nitroaniline from the chromogenic substrate included in the LAL, which is only hydrolyzed with the presence of free LPS in the sample, which activates the enzymatic cascade that leads to hydrolysis of the substrate. Using the KC4 software, the percentages of LPS recovery were obtained, which translates into the amount of free or active LPS that is not neutralized or sequestered by the evaluated peptides.
  • FICI fractional inhibitory concentration index
  • MIC A minimum inhibitory concentration of compound A
  • MIC B minimum inhibitory concentration of compound B
  • MIC AB minimum concentration of A that combined with B inhibits growth
  • MIC BA minimum concentration of B that combined with A inhibits growth.
  • Peptide A was arranged in the columns (2-11) and peptide B in the rows of the plate (B-F). In this way, 60 internal wells of the plate were used for combinations of peptide concentrations between peptide A (0.25-16 ⁇ M) and peptide B (0.075-16 ⁇ M). The first column and the last row corresponded to the concentrations of the uncombined A and B peptides, respectively.
  • 50 ⁇ L of PBS was added to all the wells of the columns and external rows of the plate. Subsequently, 50 ⁇ L of the working bacterial culture were added to all wells and the plate was incubated at 37°C for 2 h.
  • MHB double-concentrated MH broth
  • microbial growth was followed kinetically by measuring OD at 650 nm for 15 hours at 37 ° C, recording OD values at 5 min intervals on a plate spectrophotometer (BioTek Instruments, USA).
  • Bacterial growth inhibition (MIC) values were taken as values similar to the blanks (controls) of MHB medium incubated in the outermost wells of the plate. The following table shows the mean of the results obtained in three independent experiments.
  • the checkerboard method was used to evaluate the synergy between Meropenem and the peptides represented by SEQ. ID: NO. 1, SEQ. ID: NO. 2 and SEQ. ID: NO. 3 against multi-resistant bacteria Klebsiella pneumoneae and Staphylococcus aureus from clinical isolates.
  • the checkerboard method procedure used is similar to that described in Example 7. In this case, meropenem was tested as compound A and the peptides as compound B. The results shown in Table 3 are based on the determination of the Index.
  • FICI fractional inhibitory concentration

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EP21865361.6A 2020-11-19 2021-11-18 Peptide entities with antimicrobial activity against multi-drug resistant pathogens Pending EP4249500A2 (en)

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CU2020000086A CU20200086A7 (es) 2020-11-19 2020-11-19 Entidades peptídicas con actividad antimicrobiana frente a patógenos multi-drogoresistentes
PCT/CU2021/050011 WO2022105948A2 (es) 2020-11-19 2021-11-18 Entidades peptídicas con actividad antimicrobiana frente a patógenos multi-drogoresistentes

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